The number of cases of Clostridium difficile infection (CDI) has been increasing globally. CDI is the main cause of nosocomial diarrhoea, which may be life-threatening in complicated cases, and also costs the health care societies millions of pounds annually. The predominant types and their resistance to antibiotics have been changing and one of the major selective pressures which causes this is antimicrobial use. Although much is known about the role of the toxins in pathogenesis of CDI, the role of immunogenic cell wall components is unclear. They may play a role in colonisation and pathology and a study of these could clarify the infection process. It is therefore important to study the immune responses against these bacterial wall components from different strains and their effects on stimulation of leukocytes to produce cytokines and chemokines. This study was divided into four parts: 1. An epidemiological study to determine frequencies of the predominant types of C. difficile, thus 140 C. difficile isolates from surgical patients and their environment during 2009 were investigated to define their PCR ribotype. This utilised capillary sequencing gel electrophoresis for their analysis. 2. The determination of antimicrobial susceptibility to six antibiotics (ampicillin, erythromycin, tetracycline, metronidazole, moxifloxacin and vancomycin) was assessed and MIC determination by agar dilutions. 3. Investigation of host immunity to molecules with conserved molecular patterns. Surface-layer proteins (SLPs), lipocarbohydrate (LC) and flagellar proteins were separated and purified from five ribotypes of C. difficile (001, 002, 027, 078 and106) predominant in Scotland. a) The immune responses to these molecules were assessed by ELISA by exposing serum of patients and healthy donors and measuring specific IgG levels. b) Innate immunity was investigated by distinguishing responses of a macrophage cell line (THP1) to the above molecules. Induction of interleukins (IL)-1β, IL-6, IL- 8, IL-10 and IL-12 interleukins and TNF-α was detected by ELISA. In this study 15 different ribotypes were identified. The most frequent were 001, 020, 106 ribotypes (52.8%, 7.4% and 5.7%), respectively, while 13 isolates could not be assigned a ribotype. However, all isolates were sensitive to vancomycin, metronidazole and moxifloxacin, but 74.28% of isolates were resistant to erythromycin. The IgG level against bacterial antigens (SLPs, LC and flagella proteins) in donors’ serum showed almost normal distribution to all antigens from the different ribotypes and the sensitivity of the assays was increased by raising the concentration of antigens. Levels to SLPs were generally the highest, but the flagellar protein exceeded the SLPs of the 027 ribotype. The donors, controls, patients and carrier sera gave similar results. The greatest induction of interleukins was obtained using 50μg of antigen with the THP-1 cells activated with 50ng of PMA. The highest induction of all antigens was for IL-10. The highest values for the control LPS was with IL-12. But the best effect for SLPs of 027 was for IL-10 (109.1ng/ml), while the weakest for TNF for SLPs of 027 (4.7ng/ml). In general the IL-1β, IL-6, IL-8 and TNF concentrations ranged from 4.7-60ng/ml for all antigens and in contrast IL-12 and IL-10 average ranged 11- 109.1ng/ml. To conclude, the prevalence of C. difficile and their antibiotic susceptibility are constantly changing. IgG antibodies to SLPs and flagellar proteins from the hypervirulent ribotype 027 were highest in the community and hospitalized individuals. The molecules of conserved molecular patterns are immunogenic with various levels of response in the monocytic THP1 cells. SLPs were best in inducing interleukins. Flagellar proteins from 027 ribotypes accompanied SLPs in IL-10 induction levels. Consequently SLPs and flagellar proteins from 027 ribotypes appeared the best immunogenic bacterial molecules.

Class of 2009 Abstract / OBJECTIVES: The purpose of the study was to determine the rate of Clostridium difficile infections (CDI) in hospitalized patients and the various factors that were associated with the risk of developing CDI by examining patient discharge data for hospitals in 37 states in the United States using Healthcare Cost and Utilization Project (HCUP). METHODS: Patient discharge information for all patients obtained using HCUP census for the years 2002-2005, either for primary or secondary (all-listed) occurrences of CDI using the ICD-9-CM code (008.45) specific for intestinal infections due to C. difficile, were included in the study. Regression analysis, either Generalized Linear Model log-link or power-link, or a logistic regression was employed to control for the multiple independent variables. RESULTS: The incidence rate for CDI was 9.4% for the years 2002-2005. Among the concomitant diagnoses and procedures, essential hypertension, volume depletion, congestive heart failure, urinary tract infection and venous catheterization were the top 5. The length of stay (LOS) for CDI was associated with being Black, Hispanic or Other race category, number of diagnoses and procedures, primary expected payer of Medicaid, private insurance and other (including worker’s compensation, CHAMPUS,CHAMPVA etc), and all groups classified based on median household income category for patient’s zip code. Predictors of CDI related to inpatient total hospital charges were being female, race (other than black), number of diagnoses and procedures, Death, LOS, patient location and with self-pay and no charge categories as primary expected payer. Predictors of higher CDI related inpatient hospital deaths were age, female sex, Hispanic race, number of diagnoses and procedures, LOS and having Medicaid, self-pay or other as primary expected payer. CONCLUSIONS: LOS, inpatient total hospital charges, and inpatient mortality were dependent on several patient and other characteristics.

Introduction: Clostridium difficile infection (CDI) is a common hospital-acquired infection and a financial burden on the healthcare system. There is a need to reduce its impact on patients and the entire health system. More accurate estimates of the financial impact of CDI will assist hospitals in creating better CDI reduction strategies with limited resources. Previous research has not sufficiently accounted for the skewed nature of hospital cost data, baseline patient mortality risk, and the time-varying nature of CDI. Objective: We conducted a retrospective cohort study to estimate the cost impact of hospital-acquired CDI from the hospital perspective, using a number of analytical approaches. Method: We used clinical and administrative data for inpatients treated at The Ottawa Hospital to construct an analytical data set. Our primary outcome was direct costs and our primary exposure was hospital-acquired CDI. We performed the following analyses: Ordinary least square regression and generalized linear regression as time-fixed methods, and Kaplan-Meier survival curve and Cox regression models as time-varying methods. Results: A total of 49,888 admissions were included in this study (mean (SD) age of 64.6 ± 17.8 years, median (IQR) baseline mortality risk of 0.04 (0.01-0.14)). 360 (0.73%) patients developed CDI. Estimates of incremental cost due to CDI were substantially higher when using time-fixed methods than time-varying methods. Using methods that appropriately account for the time-varying nature of the exposure, the estimated incremental cost due to CDI was $8,997 per patient. In contrast, estimates from time-fixed methods ranged from $49,150 to $55,962: about a six fold difference. Conclusion: Estimates of hospital costs are strongly influenced by the time-varying nature of CDI as well as baseline mortality risk. If studies do not account for these factors, it is likely that the impact of hospital-acquired CDI will be overestimated.

Introduction: Clostridium difficile infection (CDI) is a common hospital-acquired infection and a financial burden on the healthcare system. There is a need to reduce its impact on patients and the entire health system. More accurate estimates of the financial impact of CDI will assist hospitals in creating better CDI reduction strategies with limited resources. Previous research has not sufficiently accounted for the skewed nature of hospital cost data, baseline patient mortality risk, and the time-varying nature of CDI. Objective: We conducted a retrospective cohort study to estimate the cost impact of hospital-acquired CDI from the hospital perspective, using a number of analytical approaches. Method: We used clinical and administrative data for inpatients treated at The Ottawa Hospital to construct an analytical data set. Our primary outcome was direct costs and our primary exposure was hospital-acquired CDI. We performed the following analyses: Ordinary least square regression and generalized linear regression as time-fixed methods, and Kaplan-Meier survival curve and Cox regression models as time-varying methods. Results: A total of 49,888 admissions were included in this study (mean (SD) age of 64.6 ± 17.8 years, median (IQR) baseline mortality risk of 0.04 (0.01-0.14)). 360 (0.73%) patients developed CDI. Estimates of incremental cost due to CDI were substantially higher when using time-fixed methods than time-varying methods. Using methods that appropriately account for the time-varying nature of the exposure, the estimated incremental cost due to CDI was $8,997 per patient. In contrast, estimates from time-fixed methods ranged from $49,150 to $55,962: about a six fold difference. Conclusion: Estimates of hospital costs are strongly influenced by the time-varying nature of CDI as well as baseline mortality risk. If studies do not account for these factors, it is likely that the impact of hospital-acquired CDI will be overestimated.

<p>Over three million infections were reported in the United States of America in 2019. These infections were caused by either antibiotic-resistant pathogens or <i>Clostridioides difficile</i> and resulted in more than 50,000 deaths. Unfortunately, antibacterial agents are rapidly losing their ability to treat infections and the process of discovering new antibiotics is too slow to cope up with bacterial evolution. Repurposing FDA-approved drugs of well-studied safety, pharmacology and pharmacokinetics represents a faster alternative method of antibacterial drug discovery. Repurposing is more successful and less depleting method of drug discovery than classical de novo method in regard to both cost and time. In the following studies, two major pathogens are targeted, vancomycin-resistant <i>Enterococcus</i> (VRE) and <i>C. difficile</i>. Both bacteria are more prevalent in healthcare settings were more vulnerable population of elderly and immunocompromised individuals reside. In addition, healthcare settings are usually associated with higher frequency of receiving antibiotics which in turn, compromises the integrity of normal microbiota responsible for protection against invading pathogens. Furthermore, hospital stays are associated with exposure to bacterial shedding from other patients. Our aim was to identify FDA-approved drugs with novel ability to eradicate these two bacterial pathogens in the gastrointestinal tract (GIT). Notably, the GIT is considered the actual site of infection in case of <i>C. difficile while it is only a transition site for VRE where the bacteria colonize before causing true infections in other tissues. Studies against both bacteria started with an <i>in vitro</i> screening of FDA-approved drugs and clinical molecules to identify potential candidates for further investigation.</i></p><p><i>For VRE, two drugs where identified with potent inhibitory activity and favorable pharmacokinetic profiles, auranofin and ebselen. Auranofin was approved in the 1960s for the treatment of rheumatoid arthritis due to its anti-inflammatory activity. Auranofin was found to exert potent bacteriostatic activity against both vancomycin-sensitive and vancomycin-resistant <i>Enterococcus</i> strains (minimum inhibitory concentration against 90% of the strains, MIC90 = 1 µg/mL). In addition, bacteria could not develop resistant mutants against auranofin upon prolonged exposure. On the other hand, ebselen is an organoselenium compounds currently in clinical trials for several indications. Similarly, ebselen was found to be a potent inhibitor of VRE growth (MIC90 = 2 µg/mL). In addition, ebselen successfully inhibited bacterial biofilm formation and eradicated mature biofilms. In a mouse model of VRE colonization, both drugs inhibited bacterial shedding and reduced bacterial counts in the GIT of the colonized animals.</i></p><p><i>For <i>C. difficile</i>, auranofin was also found to exert potent inhibitory activity against bacterial growth (MIC90 = 2 µg/mL), toxin production and spore formation. Additionally, it was beneficial in protecting colon cells against <i>C. difficile</i> toxin-induced inflammation. Further, auranofin was found to not promote growth of VRE as seen with the current anticlostridial agents. In addition to auranofin, two more antiprotozoal drugs were found to potently inhibit <i>C. difficile</i> growth, ronidazole and secnidazole. Both drugs are 5-nitroimidazoles approved for human (secnidazole) or veterinary (ronidazole) applications. Secnidazole and ronidazole halted <i>C. difficile</i> growth at very low concentrations (MIC90 = 0.5 and 0.125 µg/mL, respectively). Furthermore, both drugs were superior to metronidazole in bacterial killing and had favorable activities against protective gut microbiota. In addition, they demonstrated efficient protection to mice in a <i>C. difficile</i> infection model. </i></p><p><i>Overall, several drugs were presented to possess favorable activities against <i>C. difficile</i> or VRE. These drugs merit more evaluation as potential candidates for the treatment of infection caused by either bacteria. </i></p><div><i><br></i></div>